The present invention relates to phosphorus-32 and phosphorus-33 labeled proteins which are useful for radiotherapy. In particular the invention relates to methods of stably linking .sup.32 P- and .sup.33 P-containing molecules to targeting proteins in such a way that the targeting protein retains the ability to bind to a cellular target. This invention also relates to methods of therapy using the labeled proteins.
Many radionuclides have been studied for their suitability for internal administration to patients in radiotherapy. Some radionuclide compounds, containing isotopes such as .sup.131 I, can be given systemically, taking advantage of the fact that these elements tend to localize to particular tissues by virtue of their chemical properties. Other radionuclides, such as .sup.198 Au and .sup.103 Pd have been administered in a localized fashion, for instance to the site of a tumor. Most recent approaches, however, have focused on methods of delivering radionuclides to a preselected tissue by attaching the radionuclide to a targeting protein, usually an antibody, which will then localize to that tissue.
A large number of methods for linking radionuclides to antibodies have been developed. The chemical toxicity of many radionuclides means that complex methods must often be used to stably bind the isotope to an antibody. For example, to use .sup.90 Y, which has many desirable radiochemical properties, a chelate must be synthesized and covalently bound to the antibody to stably link the radioisotope to the antibody.
One isotope which displays many of the same desirable features as .sup.90 Y, but which has received little attention for targeted radiotherapy, is .sup.32 P. .sup.32 P is inexpensive, is readily available in high specific activity in a variety of labeled molecules, and has a therapeutically desirable half-life of 14 days. It is absorbed by the body and is not readily excreted, and is therefore amenable to use in outpatient procedures. In addition, .sup.32 P emits only .beta.-radiation with an excellent depth penetration in tissue of approximately 6 mm. Unlike many other radionuclides under consideration for targeted radiotherapy, it is not inherently toxic, and is currently used clinically in some non-targeted applications, for example for the treatment of ovarian cancer and polycythemia rubra vera.
Another radioisotope of phosphorus, .sup.33 P, has received even less attention than .sup.32 P. .sup.33 P shares the same chemical properties as .sup.32 P, and has similarly desirable radiochemical characteristics. It is available in high specific activity, and has a 25-day half life with a .beta.-particle emission energy of 0.25 MeV, approximately 15% of the value of the .beta.-emission energy of .sup.32 P.
The reason radioactive phosphorus has received relatively little attention for targeted radiotherapy applications has been the difficulty of linking it to targeting proteins. Most of the methods currently known are non-specific and slow, and do not efficiently incorporate radionuclide into the targeting protein.
One very general method of labeling proteins with .sup.32 P is simply to incubate the protein with .alpha.-32P-labeled nucleoside triphosphates. Schmidt et al., FEBS Lett. 194:305 (1986). The mechanism for the labeling reaction is unknown. The method is slow and gives only poor incorporation of label (less than 1% of the protein molecules are labeled), and is thus too inefficient for therapeutic use.
A second general method of .sup.32 P labeling is to incubate proteins with [.gamma.-.sup.32 P]ATP or H.sub.3.sup.32 PO.sub.4 in the presence of chromium ions. Hwang et al., Biochim. Biophys. Acta 882:331 (1986). This method is relatively rapid, but gives an unknown level of label incorporation and also leaves toxic chromium ions bound to the proteins, which would be therapeutically unacceptable.
A third general method is the use of .sup.32 P-diphenylphosphinothioic chloride as a reactive labeling compound. De Boer et al., Clin. Exp. Immunol. 3:865 (1968). This reagent is thought to react non-specifically with lysine residues in proteins to form a highly stable conjugate, but approximately 50% of the radioactivity also associates non-covalently with the labeled protein. Although this method allows labeling of proteins to high specific activity, the labeling agent is only poorly water soluble, and to achieve good labeling yields large excesses of reagent must be used, wasting relatively large amounts of hazardous radioactive materials.
A less general method of .sup.32 P labeling is the use of periodate-oxidized [.alpha.-.sup.32 P]ATP to affinity-label proteins containing an ATP-binding site. Clertant et al., J. Biol. Chem. 257:6300 (1982). Because many targeting proteins which are of therapeutic interest, in particular antibodies, do not contain ATP-binding sites this method is therefore of little general utility.
A more recent method, intended for labeling antibodies for radiotherapy, involves the chemical conjugation of protein kinase substrate peptides to antibodies. Foxwell et al., Brit. J. Cancer 57:489 (1988). The conjugates are labeled by treatment with [.gamma.-.sup.32 P]ATP in the presence of the catalytic subunit of cAMP-dependent protein kinase (protein kinase A, PKA), which transfers .sup.32 P-phosphate to a serine residue in the substrate peptide. This method showed differences in the .beta.-phase half-life between the .sup.32 P-labeled antibody and a corresponding .sup.131 -I-labeled antibody, and also high .sup.32 P uptake in the bone of animals injected with the labeled antibody. Creighton et al., "The development of .sup.32 P technology for radioimmunotherapy" in MONOCLONAL ANTIBODIES 2. APPLICATIONS IN CLINICAL ONCOLOGY. A. A. Epenetos, ed., Chapman and Hall, (1993) pp. 103-109. These results indicate in vivo instability of the label, presumably due to the action of protein phosphatases which are ubiquitous in eukaryotic cells.
It is apparent therefore, that new methods for .sup.32 P- and .sup.33 P-labeling targeting proteins are greatly to be desired. In particular, new methods in which the .sup.32 P or .sup.33 P label is stable in vivo and which do not compromise the binding abilities of these proteins are needed.